Comparative Characterization of Pseudoroegneria libanotica and Pseudoroegneria tauri Based on Their Repeatome Peculiarities

Pseudoroegneria species play an important role among Triticeae grasses, as they are the putative donors of the St genome in many polyploid species. Satellite repeats are widely used as a reliable tool for tracking evolutionary changes because they are distributed throughout the genomes of plants. The aim of our work is to perform a comparative characterization of the repeatomes of the closely related species Ps. libanotica and Ps. tauri, and Ps. spicata was also included in the analysis. The overall repeatome structures of Ps. libanotica, Ps. tauri, and Ps. spicata were similar, with some individual peculiarities observed in the abundance of the SIRE (Ty1/Copia) retrotransposons, Mutator and Harbinger transposons, and satellites. Nine new satellite repeats that have been identified from the whole-genome sequences of Ps. spicata and Ps. tauri, as well as the CL244 repeat that was previously found in Aegilops crassa, were localized to the chromosomes of Ps. libanotica and Ps. tauri. Four satellite repeats (CL69, CL101, CL119, CL244) demonstrated terminal and/or distal localization, while six repeats (CL82, CL89, CL168, CL185, CL192, CL207) were pericentromeric. Based on the obtained results, it can be assumed that Ps. libanotica and Ps. tauri are closely related species, although they have individual peculiarities in their repeatome structures and patterns of satellite repeat localization on chromosomes. The evolutionary fate of the identified satellite repeats and their related sequences, as well as their distribution on the chromosomes of Triticeae species, are discussed. The newly developed St genome chromosome markers developed in the present research can be useful in population studies of Ps. libanotica and Ps. tauri; auto- and allopolyploids that contain the St genome, such as Thinopyrum, Elymus, Kengyilia, and Roegneria; and wide hybrids between wheat and related wild species.


Introduction
The genus Pseudoroegneria (Nevski) A. Löve consists mainly of cool-season grasses that are distributed in the Middle East, central Asia, Transcaucasia, northern China, and western North America [1].Representatives of this genus are distinguished by their significant ecological plasticity and their ability to survive in arid steppe conditions [2].They also possess excellent forage quality [1,[3][4][5].Pseudoroegneria evolved 14.4-14.7 million years ago, making it more ancient than Triticum/Aegilops (8.0-8.3Myr) [6].Pseudoroegneria is represented by approximately 15 different species, including six diploids and nine autotetraploids.These species contain more than one variant of the St genome which suggests their polyphyletic origin [7,8].
At the same time, inconsistencies often occur between phylogenetic trees constructed using different genes, primarily due to incomplete lineage sorting, chloroplast captures, nuclear gene exchange through hybridization, and subsequent introgressions [8,35].These conflicts can be partially resolved by using whole-genome sequencing data as input for comparative characterization and phylogenetic analyses.With the emergence of wholegenome sequencing technologies, it has become feasible to conduct a comprehensive analysis of Triticeae genomes and determine the phylogenetics of Pseudoroegneria through a comparative analysis of the nuclear genome [5,36], chloroplast genomes [8,37], and transcriptomes [6].
Repeated elements are a reliable tool for tracking evolutionary change because they are widely distributed throughout the genome.These include both mobile elements and satellite repeats, both dispersed and tandem.They are widely used for karyotyping chromosomes, studying chromosomal rearrangements, and analyzing the genomic composition of allo-and autopolyploids and wide hybrids using fluorescence in situ hybridization (FISH) [38][39][40][41][42]. Comparative characteristics between Triticeae species can be studied by comparing the copy numbers of repeating elements [43], by comparing the distribution patterns across chromosomes and genomes [44][45][46][47], or by using a combination of both approaches [48,49].
Owing to the development of whole-genome sequencing technologies and bioinformatics analysis algorithms, it has become possible to quickly and efficiently create new chromosomal markers based on satellite repeats [50,51].
Ps. libanotica and Ps.tauri are closely related species that grow in Central Asia, specifically in Turkey, Iraq, Iran, and Syria.They are distinct from other Pseudoroegneria species as they have no awns with unequal glumes [52].The similarity of their genomes was demonstrated by analyzing chromosome pairing in interspecific hybrids [53], spectra of glutenins and gliadins [54], chloroplast and single-copy nuclear genes [55][56][57], complete chloroplast genomes [37], and Pong-like transposase sequences [58].
The comparative characteristics of closely related species are of interest for studying both the divergence of the St genome itself, which is central to a significant number of species, and for understanding the evolutionary processes within the Triticeae tribe.Here, a comparative analysis of two closely related species, Ps. libanotica and Ps.tauri, was performed by comparing their repeatomes and characterizing the chromosomal localization of newly discovered St-genome satellite repeats.
CL69.CL69 has a length of 178 bp and a 0.377% genome proportion.It shared a 98.2% identity with oligo-7E-744 from Thinopyrum elongatum, a 92.4% identity with oligo-6VS-57 from Dasypyrum villosum, an 82.4% identity with CL239 from Ae. crassa, and a 71.9% identity with CL211 from Th. bessarabicum (Table 2 and Table S1).In both studied Pseudoroegneria species, CL69 is localized terminally, but the signals appear stronger on the chromosomes of Ps. libanotica.In all fourteen chromosomes of Ps. libanotica, the signals are terminal and localized to both arms.The CL69 hybridization in Ps. tauri differs from that in Ps. libanotica not only by signal intensity but also by the absence of a hybridization site on the long arm of one chromosome (Figure 1).

Satellite Repeats Characterization and Their Chromosomal Localization in Ps. libanotica and Ps. tauri
The satellite repeats CL89, CL185, and CL192 were found in the Ps.tauri genome, while CL69, CL82, CL101, CL119, CL168, and CL207 were identified in the Ps.spicata genome.The CL244 repeat, which we had previously discovered in the Aegilops crassa genome [51], was also utilized in the experiments of in situ hybridization.For convenience, here we first describe repeats with terminal or distal localization (CL69, CL101, CL119, and CL244) and then those with mainly pericentromeric localization (CL82, CL89, CL168, CL185, CL192, and CL207).The identified repeats were submitted to the NCBI GenBank system, and the IDs OR800789-OR800793, OR800795, OR800800-OR800802 were obtained.
CL69.CL69 has a length of 178 bp and a 0.377% genome proportion.It shared a 98.2% identity with oligo-7E-744 from Thinopyrum elongatum, a 92.4% identity with oligo-6VS-57 from Dasypyrum villosum, an 82.4% identity with CL239 from Ae. crassa, and a 71.9% identity with CL211 from Th. bessarabicum (Tables 2 and S1).In both studied Pseudoroegneria species, CL69 is localized terminally, but the signals appear stronger on the chromosomes of Ps. libanotica.In all fourteen chromosomes of Ps. libanotica, the signals are terminal and localized to both arms.The CL69 hybridization in Ps. tauri differs from that in Ps. libanotica not only by signal intensity but also by the absence of a hybridization site on the long arm of one chromosome (Figure 1).CL101.CL101 has a length of 177 bp and a 0.253% genome proportion.It shared a 79.5% identity with oligo-7E-744 from Th. elongatum, a 68-76% identity with the Spelt-1 and Spelt1-similar telomeric repeats pSp1B16 and Tri-MS-6, and a 71.4% identity with CL239 from Ae. crassa (Tables 2 and S1  CL119.CL119 has a length of 668 bp and a 0.209% genome proportion.It shared a 94,7% identity with CL232 from Ae. crassa, a 90% identity with Olgo-1AL from T. aestivum, and an 84.9-89.7%identity with variants of BSCL156 from Th. bessarabicum. Additionally, identity in the range 74-86.9% was shown (in descending order) with 18-158 from Th. ponticum, CL149 from Th. bessarabicum, pAcPR5 from Agropyron cristatum, CL131 from Ae. crassa, the pTa-465 clone from Triticum aestivum, AesTR-183 from Ae. speltoides, and Sc26c38 from Secale cereale (Tables 2 and S1).In the studied Pseudoroegneria species, CL119 predominantly produces minor signals in the terminal and distal regions of most chromosomes.In two chromosomes of Ps. libanotica, intense CL119 signals are observed in the distal part of the long arm.In Ps. tauri, distinct distal CL119 signals are observed on the short arm of two chromosomes.In addition, minor signals are observed in the distal, interstitial, and proximal regions on other chromosomes in both species (Figure 1).CL244.In both Ps.libanotica and Ps.tauri, two chromosomes carry terminal hybridization sites of CL244 on the long arm (Figure 1).
CL82.CL82 has a length of 503 bp and a 0.335% genome proportion.It shared an 88% identity with the clone pTa-451 from T. aestivum and an 85% identity with CL18 from Ae. crassa and P631 from Ae. tauschii.Additionally, a lower identity (75-85%) was found for CL3 from Ae. crassa, the FAT element, oligo-5D151 from T. aestivum, StLIB98 from Ps. libanotica, oligo-7E-430 from Th. elongatum, and P523 from Ae. tauschii (Tables 3 and S2).The CL82 signals are located pericentromerically on the two chromosomes, both in Ps. libanotica and Ps.tauri (Figure 2).CL3 from Ae. crassa, the FAT element, oligo-5D151 from T. aestivum, StLIB98 from Ps. libanotica, oligo-7E-430 from Th. elongatum, and P523 from Ae. tauschii (Tables 3 and S2).The CL82 signals are located pericentromerically on the two chromosomes, both in Ps. libanotica and Ps.tauri (Figure 2).CL89.CL89 has a length of 658 bp and a 0.241% genome proportion.It shared a 100% identity with P631 from Ae. tauschii.In addition, identity in the range 75-90% was found for the pAs1 oligos and clones, P720 from Ae. tauschii, and CL3, CL193, and CL18 from Ae. crassa (Tables 3 and S2).CL89 has a similar signal distribution pattern in Ps. libanotica and Ps.tauri.Pericentromeric signals of CL89 are localized to six chromosomes of Ps. tauri and four chromosomes of Ps. libanotica (Figure 2).CL168.CL168 has a length of 476 bp and a 0.070% genome proportion.It shared a 91.7% identity with CL18 from Ae. crassa and the FAT element.A lesser degree (75-90%) was observed for P631 from Ae. tauschii, CL193 from Ae. crassa, CL80 from A. cristatum, and CL148 from Th. bessarabicum (Tables 3 and S2).CL168 is localized pericentromerically to two Ps.tauri chromosomes, and while the signal on one chromosome is bright, on the second it is minor.In Ps. libanotica, large pericentromeric signals are observed on two chromosomes, and minor pericentromeric and interstitial signals on the remaining chromosomes are visible (Figure 2).
CL185.CL185 has a length of 659 bp and a 0.033% genome proportion.It shared a 95.4% identity with P631 from Ae. tauschii and a 91.7% identity with CL18 from Ae. crassa.Additionally, identity in the range 74-83% was shown for the FAT element, CL193 from Ae. crassa, and CL148 from Th. bessarabicum (Tables 3 and S2).CL185 is a pericentromeric repeat.Bright signals were found on two Ps.libanotica and Ps.tauri chromosomes.In addition, the studied species had two additional chromosomes with less intense hybridization signals of CL185 (Figure 2).
CL192.CL192 has a length of 339 bp and a 0.029% genome proportion.It shared a 100% identity with P523 from Ae. tauschii and a 76-83% identity with Afa family repeats such as pAs1, pTa-535, and RcAfa (Tables 3 and S2).CL192 is present in both species.The signals are located pericentromerically on two chromosomes (Figure 2).
CL207.CL207 has a length of 657 bp and a 0.028% genome proportion.It shared a 90.5% identity with CL18 from Ae. crassa and the FAT element (Tables 3 and S2).Both studied species have pericentromeric localization sites of CL207, but the signal intensity varies among chromosomes.In Ps. libanotica, three chromosomes have bright signals, and three chromosomes have less intense localization sites.Ps. tauri is characterized by the presence of two chromosomes with strong pericentromeric signals of CL207 and four chromosomes with fainter signals (Figure 2).

Discussion
Studying the repeatome in wild grasses is important for understanding the processes of speciation.In total, the structure of the repeatome and the percentage of different lineages of mobile elements in Ps. libanotica were very similar to those revealed in [50].According to the analysis of the whole-genome sequences, the number of PIF/Harbinger reads in Ps. tauri was 1.4 times larger than that in Ps. libanotica (Table 1), which agrees with the data obtained from the copy number of Pong (belonging to PIF/Harbinger) [58].According to Markova et al. (2015), the abundance of PIF/Harbinger is equal in Ps. spicata and Ps.tauri [58].However, according to our data, Ps. spicata has 6 and 10.5 times fewer PIF/Harbinger reads compared to Ps. libanotica and Ps.tauri, respectively, which can probably be explained by the different accessions of Ps. spicata.In our previous study, we found that Ps. spicata Angela showed an overwhelming majority among the studied transposons of the Ty1/Copia family [43], which is consistent with the findings of this study.In the genome of Ps. libanotica, it had almost twice as many satellite sequences, while the genome of Ps. tauri showed a higher proportion of the Athila element.Thus, although the overall structure of the repeatome between these two Pseudoroegneria species is similar, there are also some differences.
Satellite repeats can be used to create chromosomal markers that enable a comparative analysis between species, establishing the degree of their genetic similarity.Among the nine repeats localized to the Ps.libanotica and Ps.tauri chromosomes, four (CL69, CL101, CL119, CL244) showed predominantly terminal and/or distal localization (Figure 1), while six showed mainly pericentromeric localization (CL82, CL89, CL168, CL185, CL192, CL207) (Figure 2).The predominant localization in pericentromeric and/or terminal repeats is characteristic of non-dispersed repeats identified in the St genome, as described in the literature.Terminal localization on the chromosomes of the St genome is typical for the St-96 and St-98 repeats from Ps. libanotica [50], St 2 -80 and pPlTaq2.5 from Ps. libanotica [45,59], and S159 from Ps. stipifolia [47].Pericentromeric localization has been shown for CentSt, Plants 2023, 12, 4169 8 of 15 S17, and S170 from Ps. stipifolia [47,49].STlib_117 signals from Ps. libanotica were visible in the centromeric and terminal regions [50].Interestingly, the repeats identified in the present study did not show any similarity to any of the previously published repeats found in the St genome.CL69 signals were observed on all the chromosomes in the terminal regions of Ps. tauri and Ps.libanotica (Figure 1).Repeats similar to CL69 also showed predominantly telomeric localization in Triticeae species (Tables 2 and S1), such as CL239 from Ae. crassa on the chromosomes of Ae. crassa and Th.bessarabicum [51], oligo-6VS-57 from D. villosum on the chromosomes of D. villosum [60], and oligo-7E-744 from Th. elongatum on the chromosomes of D. villosum and D. breviaristatum, as well as on the St chromosomes of E. dahuricus [61,62].Thus, the conservation and ancient origin of the listed repeats and CL69 can be assumed to stem from a common ancestral repeat.
CL101 signals of varying intensity were observed on three pairs of Ps. tauri chromosomes, but they were not detected in Ps. libanotica (Figure 1).The similarity of CL101 to other repeats found in the species of Aegilops, Triticum, Elytrigia, and Dasypyrum may also indicate its ancient origin.At the same time, the percentage identity with the oligo-7E-744, pSp1B16, CL239, and Spelt1 repeats did not exceed 80%.The chromosomal distribution of the CL101 homologues across the Triticeae genomes includes both terminal and interstitial localization [51,[61][62][63][64]. Therefore, CL101 and its related repeats have a different evolutionary fate and distribution among species and chromosomes.
The strongest distal CL119 signals were observed in Ps. libanotica and Ps.tauri on one pair of chromosomes, and minor signals were observed in various regions of the remaining chromosomes (Figure 1).The localization of the CL119-like repeats in Triticeae species is characterized by distal, subtelomeric, and terminal localization on chromosomes (Tables 2 and S1) [51,[63][64][65][66].These repeats have also been found on B chromosomes of rye and Aegilops [67,68], except for pAcPR5, which is distributed across all P genome chromosomes of A. cristatum [69].It may be noted that both CL119 and similar repeats predominantly produce the strongest signals on one or more pairs of chromosomes, including B chromosomes.This may suggest their role in the specificity of chromosome recognition during cell division.
The CL244 repeat used in this study was previously found in the genome of Ae. crassa [51].Ps. tauri and Ps.libanotica exhibited a similar type of hybridization, occurring terminally on the long arm of one pair of chromosomes (Figure 1).In our previous study, CL244 hybridized terminally on several chromosome pairs of Ae. crassa, T. aestivum, and Th.bessarabicum, while in the latter species, the signals were the strongest.Given the conserved nature of localization and its distribution in many species of Triticeae, as well as the similarity of the CL244 terminal repeat to the Spelt52.1 repeats from Ae. Speltoides [70], pSc200 and pSc7235 from S. cereale [71,72], and BSCL1 and DP4J27982 from Th. bessarabicum [66,73], it can be assumed that CL244 refers to ancient repeats that arose before the divergence of the hypothetical ancient genome into separate genomes.
All six pericentromeric repeats showed homology to the FAT repeat (Tables 3 and S2).Most often, the FAT element exhibits "fuzzy hybridization" with greater hybridization in the proximal and pericentromeric regions of the D genome chromosomes in T. aestivum, as well as on the chromosomes of the C, D, N, M, S, and U genomes in various Aegilops species [74].The FAT repeat on Ps. spicata chromosomes shows a dispersed pattern in the proximal region, with the most intense signal observed in one pair of chromosomes [46].Furthermore, all the pericentromeric repeats identified in the current study, with the exception of CL192, exhibited similarity to the CL18 repeat from Ae. crassa.CL18 exhibited an uneven distribution along the length of the chromosomes of Ae.Crassa, Th.Bessarabicum, T. aestivum, and Ae.tauschii, with more intense hybridization in the proximal chromosome regions [51].The same five repeats showed homology to ACRI_CL80, which is localized pericentromerically to the A. cristatum chromosomes [75].Four pericentromeric repeats, CL168, CL82, CL185, and CL89, showed homology to the pericentromeric repeat P631, which we previously found in the genome of Ae. tauschii and is characterized by either a discrete pericentromeric signal in Th. bessarabicum, Th. intermedium, and Ps.spicata or dispersed with strong pericentromeric signals in wheat and rye chromosomes [76,77].This difference in hybridization patterns can be explained by the occurrence of these sequences in a common ancestor in the pericentromeric region of Triticum, Aegilops, Thinopyrum, Secale, and Pseudoroegneria.The number and distribution of elements have changed during subsequent evolution, resulting in variations in hybridization patterns.Although the listed repeats are homologous to each other, some of them are dispersedly spread from the pericentromeric region to the proximal regions, such as FAT and CL18.Others are localized in the pericentromeric region, like the six repeats we found and ACRI_CL80.Additionally, some repeats, such as P631, exhibit unique distribution patterns across different species.
It is worth noting that although CL89 (658 bp) is 100% identical to P631 (317 bp) (Table 3), it has a greater length (Table 4).Similarly, the pericentromeric repeat CL192 (339 bp) is 32% smaller in size than the P523 repeat (501 bp), which we previously identified in the genome of Ae. tauschii and is localized pericentromerically in the Js chromosome pair of Th. intermedium [76].Thus, 100% identity in these cases indicates the proximity of these repeats, but not a perfect match.
The repeats CL89, CL82, and CL192 were found to be similar to Afa family repeats such as pAs1 and pTa535 from T. aestivum [78], RcAfa from Roegneria ciliaris [79], and CL3 from Ae. crassa [51] (Table 3).The Afa family is commonly used for chromosome identification in the Triticeae tribe and typically results in the detection of multiple subtelomeric, proximal, and interstitial hybridization sites on chromosomes [44,51,73,80].CL82, CL89, and CL192 showed only pericentromeric signals in Ps. tauri and Ps.libanotica (Figures 1 and 2).Despite the sequence's proximity to the Afa family, the localization pattern of the repeats presented here is significantly different from that of the Afa family.This difference may indicate a divergence of CL192 from the ancestral form that is common to the Afa family.
The comparison of the localization of the identified repeats on the chromosomes of Ps. libanotica and Ps.tauri provides the following classification.
(ii) Repeats with a similar pattern of hybridization with some differences: CL69, CL207, and CL168.
(iii) Repeats with different patterns of hybridization, exhibiting variations in the number of chromosomes or hybridization sites: CL119, CL101, and CL89.
Thus, based on this classification and a comparison of the repeatome structure, we can conclude that Ps. libanotica and Ps.tauri are distinct, closely related species, each with unique patterns of satellite repeat distribution and distribution along chromosomes.This conclusion is supported by the fact that both studied species cluster together in molecular genetic studies and share similar morphological characteristics [52][53][54][55][56][57][58].The chromosomal markers we have created could be valuable for conducting population studies of these species, as well as for evaluating their biodiversity and speciation.Notably, the brightest signals are CL101 and CL168 in Ps. tauri and CL207 in Ps. libanotica, which were observed on an odd number of chromosomes, which is typical for cross-pollinated species with a heterozygous genome [40,51].Among the three groups presented, the second and third groups may be the most suitable for such studies, as they exhibited differences among the studied Pseudoroegneria species.From this perspective, the satellite repeats revealed here can be utilized to determine the evolutionary status among different Pseudoroegneria accessions.For this purpose, the developed chromosome markers are to be precisely localized to specific linkage groups using bulked Oligo-FISH, which is based on a mixture of single-copy sequences [84].The St genome chromosome markers developed in the present research can be useful in studies of polyploid species that contain the St genome, such as Thinopyrum, Elymus, Kengyilia, and Roegneria, as well as in wide hybrids.

Plant Materials
The following plant material was used in the study: Ps. libanotica PI 228389, Ps. tauri PI 380652, and Ps.spicata PI 578855.All accessions are diploids with the genomic formula StSt and were kindly provided by the USDA-ARS Germplasm Resources Information Network (GRIN).

Sequencing and Bioinformatics Analysis
Genomic DNA was isolated by the CTAB protocol [79].The quality and quantity of the isolated DNA were tested using Qubit 4 (Thermo Fisher Scientific, Waltham, MA, USA) and electrophoresis in an 0.8% agarose gel.
Shotgun sequencing libraries were synthesized using the Swift 2S Turbo DNA Library Kit (Swift Bioscience, Ann Arbor, MI, USA), and their quality was checked using MiSeq (Illumina, Inc., San Diego, CA, USA).Already converted bible libraries were sequenced on the DNBSEQ-G400 device (MGI Tech, Shenzhen, China).The initial amount of DNA used was 25 ng, and fragments of about 350 bp in size were indexed at both ends using the Swift 2S Turbo Unique Dual Indexing Kit (Swift Bioscience, USA).Sequencing was performed on Illumina NextSeq (Illumina, Inc., San Diego, CA, USA) using the NextSeq 500/550 Mid Output Kit v2.5 (llumina, Inc., San Diego, CA, USA).
The subsequent study of nucleotide sequences, the search for repetitive DNA sequences, and the identification of their uniqueness were carried out in accordance with the methodology described in [45].The sequences of primers for the identified satellite repeat monomers are shown in Table 4.

Fluorescence In Situ Hybridization (FISH)
The fixation of the material and the preparation of cytological preparations from the root meristems were performed in accordance with the methodology presented in the article [80].The probes were localized on Ps. libanotica and Ps.tauri chromosomes using flu-
). Six chromosomes of Ps. tauri carry terminal signals of CL101: four chromosomes showed signals on the short arm, while two chromosomes showed signals on the long arm.The strongest signal is observed on one chromosome, Plants 2023, 12, 4169 5 of 15 while the rest are very faint.In the chromosomes of Ps. libanotica CL101 signals are absent (Figure 1).

Table 2 .
Results of the homology search for new St-genome terminal satellite repeats with known Triticeae repeats.
* data is not available; ** no homology was revealed.

Table 3 .
Results of the homology search for new St genome pericentromeric satellite repeats with known Triticeae repeats.

Table 3 .
Results of the homology search for new St genome pericentromeric satellite repeats with known Triticeae repeats.
* data is not available; ** no homology was revealed.

Table 4 .
Primer sequences for the tandem repeats.